A number of nucleoside analogs such as cytarabine, fludarabine, cladribine, capecitabine, gemcitabine and pentostatin are used clinically as highly effective anti-neoplastic agents. Among these, gemcitabine (2′,2′-difluoro-2′-deoxycytidine; marketed as Gemzar™) is of particular interest due to its unique activity against solid tumors. It is currently approved to treat breast, non-small cell lung, ovarian and pancreatic cancers and widely used to treat a variety of other cancers including bladder, biliary, colorectal and lymphoma.
Several self-potentiating mechanisms unique to this nucleoside analog are believed responsible for the activity of gemcitabine against solid tumors. The diphosphate metabolite of gemcitabine inhibits ribonucleotide reductase, which results in lower concentrations of intracellular deoxycytidine triphosphate (dCTP) and thus, increased incorporation of the triphosphate gemcitabine metabolite into DNA, which results in inhibition of DNA synthesis and blocks completion of the cell division cycle. Additionally, reduction in dCTP concentration up regulates the enzyme cytidine kinase, which is responsible for initial phosphorylation of gemcitabine, a necessary step in the inhibition of DNA synthesis by the drug. Finally, the triphosphate metabolite of gemcitabine is an inhibitor of cytidine deaminase, which is responsible for gemcitabine inactivation by conversion to the uridine metabolite. Accordingly, the additive nature of the above factors may explain the efficacy of gemcitabine in treating solid tumors.
Due to the lipophilic nature of the ProTides, these molecules can deliver nucleoside monophosphates directly in to the intact tumor cell. Previous studies have characterized multiple cellular transport mechanisms for nucleoside analog drugs and their derivatives (for a review, see Balimane et al., Adv. Drug Delivery Rev. 1999, 39, 183-209). A relatively hydrophilic compound, gemcitabine has limited ability to permeate plasma membranes via passive diffusion and several studies have demonstrated that gemcitabine is a substrate for equilibrative and concentrative nucleoside transporters (ENT's and CNT's respectively). Specifically, gemcitabine is transported by human ENT1, ENT2, CNT1 and CNT3, but not the purine-selective concentrative transporter CNT2 (see Mackey et al., Cancer Res. 1998, 58, 4349-4357; Mackey et al., J. Natl. Cancer Inst. 1999, 91, 1876-1881; and Fang et al., Biochem. J. 1996, 317, 457465).
U.S. Pat. No. 4,808,614 discloses 2,2′-difluoronucleosides which are known anti-viral and anti-tumor agents, in particular 2′,2′-difluoro-2′-deoxycytidine (commonly known as Gemcitabine).
U.S. Pat. No. 7,951,787 discloses phosphoramidate derivatives of nucleotides such as 2′-deoxy-2′,2′-difluoro-D-cytidine-5′-O-[phenyl(benzoxy-L-alaninyl)] phosphate (also referred to as Gemcitabine-[phenyl(benzoxy-L-alaninyl)] phosphate or NUC-1031). Methods for chemically synthesizing these derivatives are disclosed in this patent by reacting gemcitabine or its structural variants with a diastereoisomeric mixture of phosphochloridate such as phenyl-(benzoxy-L-alaninyl)-phosphorochloridate of Formula II in the presence of N-methylimidazole followed by purification of the product by column chromatography, eluting with dichloromethane/methanol 95:5 to give pure product as a white foamy solid with very low yield of 16%.

NUC-1031 is typically prepared as a mixture of two diastereoisomers, epimeric at the phosphate centre. The diastereoisomers of NUC-1031 have the following structures:

NUC-1031 is extremely lipophillic and thus poorly water soluble (by calculation: <0.1 mg/mL), and the ionisable moieties have calculated pKas which lie out-side the pH range suitable for parenteral administration. It is essentially insoluble in water, regardless of salt content or pH, and this has implications for the development of formulations for delivering the compound at sufficiently high dosages for effective treatment. It also has implications for the development of efficient manufacturing processes which will allow NUC-1031 to be produced cost effectively.
It has recently been discovered that the (S)-epimer of gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate has sufficient solubility in mixtures of a number of polar organic solvents with water to render it suitable for formulation and administration as a therapeutic agent. The solubility of the (R)-epimer is considerably lower. In certain solvent mixtures the difference in solubility between the (S)-epimer and the (R)-epimer is over 100 fold. It is expected therefore that more clinically effective, practical and patient friendly administration methods can be developed using the (S)-epimer than can be developed using the (R)-epimer or using the mixture. It is thus desirable to be able to provide gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(S)-phosphate in substantially diastereoisomerically pure form.
The low solubility of NUC-1031 in many solvents, particularly those commonly used in separating compounds using HPLC, mean that large volumes of solvent would be needed for any HPLC based separation. This means that any HPLC based industrial scale separation process would be high cost, consume large amounts of energy and material and produce large amounts of waste.
Although it appears preferable at the time of filing this application to administer gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate as the (S)-epimer, one can also conceive of reasons for needing to obtain the (R)-epimer in a diastereoisomerically pure form. These would include the carrying out of comparative tests, to convert the (R)-epimer to the (S)-epimer or because the (R)-epimer provides benefits over the (S)-epimer which outweigh its low solubility.
Indeed the (R)-epimer has been shown to have a half-life on incubation with isolated human hepatic cells which is four times that of the (S)-epimer. The longer half-life associated with (R)-isomer indicates a lower intrinsic clearance and should result in a different pharmacokinetic and pharmacodynamic profile to the (S)-isomer which may offer some benefits.
Both (S)- and (R)-epimers are therapeutically active.
WO 2014/076490 discloses a process for preparation of nucleoside prodrugs such as Gemcitabine-[phenyl(benzoxy-L-alaninyl)] phosphate by reacting gemcitabine or its structural variants with a diastereoisomeric mixture of phosphochloridates in the presence of a catalyst comprising metal salt such as Cu(OTf)2, CuCl, CuBr, CuI, Cu(OAc)2, CuSO4, Cu(OC(O)CF3)2, Me(OTf)3, Cu(OTf)2, Yb(OTf)3, Fe (OTf)3, La(OTf)3 with yield of 45%.
It is an aim of certain embodiments of this invention to provide a method of providing gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(S)-phosphate in substantially diastereoisomerically pure form.
It is an aim of certain embodiments of this invention to provide a method of providing gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(R)-phosphate in substantially diastereoisomerically pure form.
It is an aim of certain embodiments of this invention to provide a method of providing the (S) and/or (R)-epimer(s) in substantially diastereoisomerically pure form(s) which is scalable, economic and/or efficient, e.g. more scalable, economic and/or efficient than methods using HPLC. Thus, it is an aim of certain embodiments of this invention to provide a method of providing the (S) and/or (R)-epimer(s) in substantially diastereoisomerically pure form(s) which is suitable for large scale manufacture.
It is an aim of certain embodiments of this invention to provide a simple method i.e. a method which involves a minimum number of process steps and or reagents of providing the (S) and/or (R)-epimer(s) in substantially diastereoisomerically pure form(s).
Another aim of certain embodiments of this invention is to provide a method which ensures the separated (S)- or (R)-epimer are provided in substantially diastereoisomerically pure form and at the same time meet or exceed the necessary criteria stipulated by organisations such as the US FDA concerning the amounts and nature of any trace impurities which arise from synthesis and separation.
Certain embodiments of this invention satisfy some or all of the above aims.